Genomic instability is a hallmark of cancer cells. The spindle checkpoint ensures genomic integrity by inhibiting anaphase onset if even a single chromosome is not properly aligned on the spindle. Recent evidence suggests that defects in mammalian homologues of the yeast checkpoint genes may contribute to the generation of human cancer. The long-term objective of this application is to understand the molecular mechanisms that coordinate cell cycle processes to ensure accurate cell division in mammalian cells. The specific aims of this proposal are: (1) to determine if an intrinsic timing component contributes to proper order of the cell cycle, (2) to determine if checkpoint proteins are essential for normal cell division, and (3) to determine the molecular interaction among checkpoint proteins and regulations of anaphase. These aims will be addressed in cultured cells with live cell imaging in combination with in vitro studies using immunoprecipitation and affinity chromatography. Chromophore- assisted-laser inactivation (CALI) of immunoprecipitation and affinity chromatography. Chromophore-assisted-laser inactivation (CALI) of protein function will be used to produce "cellular knockouts" of checkpoint proteins. The focus of the protein function will be used to produce "cellular knockouts" of checkpoint proteins. The focus of the biochemical studies will be molecular interactions of human Mad1 with other checkpoint proteins and regulators of anaphase. These studies will contribute to our understanding of the mechanisms employed by mammalian cells to prevent inaccurate chromosome segregation and maintain genomic integrity.